The beta2-adrenergic receptor (beta2AR) is one of the most targeted seven transmembrane G protein-coupled receptors for drug development and for molecular studies, largely because of the prevalence of asthma in the population, the symptoms of which are treated with drugs that specifically activate the receptor leading to brochodilation. Acute drug stimulation of the beta2AR in cells leads to a rapid desensitization (min) and chronic stimulation (days) to even further loss of its activity, thus reducing the effectiveness of therapy. Over the past several decades we and others have shown that the mechanisms of rapid agonist-induced desensitization clearly involve cAMP-dependent protein kinase (PKA) and G protein receptor kinase (GRK) phosphorylation of the beta2AR, arrestin binding, and trafficking of the beta2AR from the cell surface to endosomes. The slow downregulation is in part correlated with the rapid phosphorylation events but also involves proteolysis of the beta2AR and inhibition of beta2AR synthesis. One of the limitations of previous studies was the inability to directly measure the phosphorylations of residues implicated in beta2AR desensitization. To address this we developed several phosphosite-specific antibodies to key residues and used them to directly characterize the kinetics of PKA and GRK phosphorylations for the first time in human embryonic kidney (HEK) 293 and epidermoid carcinoma A431cell lines. In this proposal we intend to further develop the phospho-specific antibody approach to determine the kinetics of putative as well as known GRK and PKA residue phosphorylations. We will address the following hypotheses: (i) that agonists stimulate individual GRK and PKA site phosphorylations on the beta2AR at different rates, and that those relative rates may be altered by the particular agonist used; (ii) that the phosphatases involved in dephosphorylation of PKA sites differ both in locale and subtype from dephosphorylation of GRK sites; (iii) that multiple GRKs phosphorylate the beta2AR in a site- and cell type selective manner; (iv) that peptides that mimic the beta2AR GRK sites can be effective substrates/inhibitors of GRK phosphorylation in cells. Finally we will characterize the mechanisms of agonist-induced beta2AR phosphorylation, internalization, recycling and downregulation in primary cultures of human lung smooth muscle cells, with an emphasis on agonists used for the treatment of asthma.